CN114057881B - Anti-ketamine specific antibodies, plasmid vectors and methods - Google Patents
Anti-ketamine specific antibodies, plasmid vectors and methods Download PDFInfo
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- CN114057881B CN114057881B CN202111626212.7A CN202111626212A CN114057881B CN 114057881 B CN114057881 B CN 114057881B CN 202111626212 A CN202111626212 A CN 202111626212A CN 114057881 B CN114057881 B CN 114057881B
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Abstract
According to the application, a mouse is immunized by using a KET-BSA conjugate, spleen lymphocytes of the mouse are separated, B lymphocytes which are specifically combined with the KET-BSA conjugate are separated by using a flow cytometry, antibody heavy chain and light chain variable region sequences in the B lymphocytes are amplified by using single cell PCR, the obtained sequences are constructed into a complete mouse IgG antibody sequence expression vector, a monoclonal antibody is expressed by transient HEK293F cells, the monoclonal antibody is purified, and dominant monoclonal antibody is screened by a colloidal gold chromatography experiment, so that the time is greatly shortened compared with the preparation of the traditional monoclonal antibody, and the obtained monoclonal antibody has high stability and good uniformity, and the inter-batch difference is greatly reduced.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to an anti-ketamine specific antibody, a plasmid vector and a method.
Background
Ketamine, K powder, has serious dependence, addiction is more concealed than traditional drugs, entertainment often covers the addiction, and initial psychological dependence is formed gradually after the frequency and the contact times are increased. Chronic abuse of ketamine can harm urinary system, central nervous system, cardiovascular system, respiratory system, digestive system, memory loss, cognitive impairment and psychosis. Ketamine engulfs people's health just like tiger, and ketamine is listed as a first class of psychotropic drugs in China by most countries of the world. Therefore, the research of specific antibodies capable of being used for ketamine detection is particularly important.
Disclosure of Invention
In order to achieve the above design objective, according to a first aspect of the present application, there is provided an anti-ketamine specific antibody comprising a light chain and a heavy chain, wherein the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 1; the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.1 is shown as SEQ ID NO. 5; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.2 is shown as SEQ ID NO. 6.
In a second aspect of the present application, there is provided a plasmid vector comprising the light chain variable region nucleotide sequence shown as SEQ ID NO. 5.
In a third aspect of the present application, there is provided a plasmid vector comprising a heavy chain variable region nucleotide sequence as set forth in SEQ ID NO. 6.
In a fourth aspect of the application, there is provided an anti-ketamine specific antibody comprising a light chain and a heavy chain, wherein the variable region amino acid sequence of the light chain is shown in SEQ ID NO. 3; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 4.
Further, the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.3 is shown as SEQ ID NO. 7; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.4 is shown as SEQ ID NO. 8.
In a fifth aspect of the present application, there is provided a plasmid vector comprising the light chain variable region nucleotide sequence shown as SEQ ID NO. 7.
In a sixth aspect of the present application, there is provided a plasmid vector comprising a heavy chain variable region nucleotide sequence as set forth in SEQ ID NO. 8.
In a seventh aspect of the present application, there is provided a method for eukaryotic expression of an anti-ketamine specific antibody plasmid vector, comprising the steps of:
a) The nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region are respectively bridged with the nucleotide sequence of the mouse IgG light chain constant region and the nucleotide sequence of the heavy chain constant region by PCR, and then are respectively connected with plasmid vectors to construct eukaryotic cell expression vectors;
b) Transfecting the eukaryotic expression vector in the step a) to HEK293F cells to express to obtain an anti-Ketamine (KET) monoclonal antibody;
c) Purifying the monoclonal antibody, and determining dominant monoclonal antibody through a colloidal gold chromatography experiment;
the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.5 or as SEQ ID NO. 7;
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.6 or SEQ ID NO. 8.
The application has the advantages that: an anti-ketamine specific antibody is provided.
Specific embodiments:
although the following embodiments describe the design concept of the present application in more detail, these descriptions are merely descriptions of the design concept of the present application, and not limitations of the design concept of the present application, and any combination, addition or modification of the design concept of the present application will fall within the scope of the present application.
Preparation of Ketamine (KET) monoclonal antibody
Female Balb/c mice of 4-6 weeks old were given, and each was immunized subcutaneously with multiple injections of 100. Mu.g KET-BSA emulsified with Freund's complete adjuvant, 400. Mu.l/mouse total. Boosting was performed 20 days later by taking 400 μl/min of 80 μg recombinant protein emulsified with Freund's incomplete adjuvant and performing subcutaneous multipoint injection. After 15 days of the third boost, the procedure was the same as the second boost. After 20 days, 120. Mu.g of KET-BSA was injected intraperitoneally, mice were sacrificed after 72 hours, spleens of the mice were removed, spleen lymphocytes of the mice were isolated by using a mouse lymphocyte separation kit (Tianjin, ocean biologicals science and technology Co., ltd.), target B lymphocytes were stained with a fluorescent-labeled probe prepared by KET-BSA, and single B cells expressing specific antibodies were isolated therefrom by means of a flow cytometry sorting technique. mRNA of single B cell is extracted, RT-PCR is performed to synthesize cDNA, cDNA is taken as a template, universal degenerate primers of a murine antibody variable region are adopted to amplify nucleotide sequences of a light chain variable region and a heavy chain variable region of an encoded antibody respectively, the nucleotide sequences are respectively connected with a constant region, and then the nucleotide sequences are inserted into a pcDNA3.1 (+) vector to construct recombinant plasmids for expressing specific antibody light chains and heavy chains. The light chain plasmid and the heavy chain plasmid of the same antibody are mixed according to the following ratio of 1:1 mass ratio, and then transfected into HEK293F cells for expression and assembly of light and heavy chains of monoclonal antibodies, and collecting cell culture liquid and purifying the monoclonal antibodies by Protein A affinity. Purity was checked by SDS-PAGE electrophoresis. The following day was subjected to monoclonal ELISA screening, the screening steps were as follows:
coating: respectively diluting KET-BSA conjugate and BSA to a concentration of 1 mug/mL with a coating solution, adding an ELISA plate (Shenzhen Jin Canhua Co., ltd.) into 100 mug/hole, and washing with a washing solution for 1 time by a DEM-3 plate washer (Zhongshan university Daan Gene Co., ltd.) after overnight at 4 ℃;
closing: adding a sealing liquid at 200 mu L/hole, sealing for 2 hours at 37 ℃, and washing 1 time by using a washing liquid through a plate washer;
sample adding: diluting the purified antibody according to a certain proportion, respectively adding KET-BSA conjugate and BSA coated holes, incubating for 1h at 37 ℃ with 100 mu L/hole, and washing 3 times by using a washing liquid through a plate washer;
adding enzyme-labeled antibody: fresh diluted HRP enzyme-labeled secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) was added to the KET-BSA conjugate and BSA coated wells at 100. Mu.L/well, and after incubation at 37℃for 30 minutes, washed 4 times with washing liquid by a plate washer;
adding a color development liquid: adding 50 mu L of each of the developing solution A and the developing solution B into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
terminating the reaction: adding a stop solution at a concentration of 50. Mu.L/well;
and (3) result judgment: on a microplate reader, OD values were read after blank wells were zeroed at 450 nm. The serum of immunized mice was used as a positive control. The results showed that there were 4 positive clones with higher OD in the KET-BSA conjugate wells, no color development in the BSA wells, and 4 sequences were obtained by sequencing, 4A1,6B2,7C3,6D4, respectively.
The relevant solution formulation is as follows:
coating liquid: na (Na) 2 CO 3 1.5g，NaHCO 3 2.9g, add ddH 2 O was fixed to 1000mL (pH 9.6).
Sealing liquid: na (Na) 2 HPO 4 .12H 2 O 2.68g，NaH 2 PO 4 .2H 2 O0.39g,NaCl 8.5g,20g bovine serum albumin, ddH 2 O was fixed to 1000mL (pH 7.4).
Washing liquid: na (Na) 2 HPO 4 .12H 2 O 2.68g，NaH 2 PO 4 .2H 2 O0.39g,NaCl 8.5g,Tween-20.5 mL, add ddH 2 O was fixed to 1000mL (pH 7.4).
Color development liquid A:200mg TMB is dissolved in 100mL absolute ethanol, add ddH 2 O is fixed to 1000mL.
Color development liquid B: citric acid 2.1g, na 2 HPO 4 .12H 2 O71 g, add ddH 2 O is fixed to 1000mL.
When in use, the utility model is characterized in that: 1mL of color developing solution A+1mL of color developing solution B+0.4. Mu.L of 30% H 2 O 2
Stop solution: 2M H 2 SO 4 21.7mL of concentrated H 2 SO 4 Adding ddH 2 O is fixed to 1000mL.
Preparation of colloidal gold pad
Taking 5ml of 0.01% colloidal gold solution, adding 10 mu L of 0.2mol/L potassium carbonate solution, fully mixing, adding 50 mu g of monoclonal antibody, uniformly mixing, standing at room temperature for 2 hours, adding 500 mu L of 10% BSA (bovine serum albumin) solution for sealing, centrifuging (10000 rpm/min, 20 min) after sealing treatment for 2 hours, and fully dissolving the sediment by 500 mu L of complex solution after discarding the supernatant. The dissolved gold solution was uniformly sprayed on a 6mm wide glass fiber with a gold spraying and film drawing instrument (Sangzhou Houzan technologies Co., ltd.) at a concentration of 6. Mu.l/cm, and then was dried by air blowing at 37℃in an electric heating air drying oven (Shanghai-Heng science instruments Co., ltd.) for 2 hours.
The relevant solution formulation is as follows:
0.01% colloidal gold solution: 1ml of 1% chloroauric acid solution and 1.4ml of 1% citric acid solution, and adding ultrapure water to heat and dissolve the mixture, and fixing the volume to 100ml.
1% chloroauric acid solution: auCL (AuCL) 3 .HCl.4H 2 O powder 1g was dissolved in ultrapure water and the volume was fixed to 100ml.
1% citric acid solution: 1g of citric acid crystal was dissolved in ultrapure water and the volume was fixed to 100ml.
0.2mol/L potassium carbonate solution: 27.64g of potassium carbonate was dissolved in ultrapure water and the volume was set to 1000ml.
And (3) a complex solution: tris base 6.057g was dissolved in 800ml of ultrapure water, the pH was adjusted to 8.0 with an appropriate amount of HCL, and the volume was adjusted to 1000ml by adding ultrapure water.
Preparation of nitrocellulose Membrane (NC Membrane)
After ketamine-BSA was diluted with coating liquid (final concentration: 1 mg/ml), it was uniformly coated on a nitrocellulose membrane (Sartorius) by a metal spraying and film drawing instrument (Hangzhou brand and technology Co., ltd.) at 1. Mu.l/cm, which is a T line; sheep anti-mouse solution (final concentration 1 mg/ml) was uniformly coated on nitrocellulose membrane at 1. Mu.l/cm, which was line C. After the film coating was completed, the nitrocellulose film was dried at 37℃for 12 hours in an electrothermal forced air drying oven (Shanghai-Heng scientific instruments Co., ltd.).
Preparation of colloidal gold immunity detection card
Assembling a test strip: sequentially overlapping and pasting on a PVC bottom plate: (1) NC membrane coated with KET-BSA as detection area and goat anti-mouse IgG as quality control area; (2) A gold pad sprayed with a colloidal gold-labeled Ketamine (KET) monoclonal antibody (4A1,6B2,7C3,6D4); (3) The sample pad is a glass fiber membrane treated by 2% Tween-20; (4) And (3) cutting the water absorbing paper into a width of 4mm after the assembly is completed, mounting a reagent card strip shell, and compacting to obtain the colloidal gold immunochromatography detection card.
Monoclonal antibody screening
The positive urine sample and the normal urine sample are loaded in 80 mu L/hole, and after being placed at room temperature for 15min, the positive urine sample and the normal urine sample are respectively read by a colloidal gold colorimetric card, and the results are shown in Table 1 in detail.
TABLE 1 colloidal gold colorimetric card value statistics
From the above table, it can be seen that the 4A1 mab and the 6B2 mab are 2 dominant mab strains for detecting Ketamine (KET).
According to the application, a mouse is immunized by using a KET-BSA conjugate, B lymphocytes which are specifically combined with the KET-BSA conjugate are separated by a flow cytometry, the heavy chain and light chain variable region sequences of antibodies in the lymphocytes are amplified by using single-cell PCR, and the obtained sequences are constructed into a monoclonal antibody of the whole mouse IgG antibody expression vector expression anti-Ketamine (KET), and the monoclonal antibody is applied to the field of drug detection.
The application aims to solve the defects of the traditional preparation of monoclonal antibodies, the KET-BSA conjugate is used for immunizing mice, B lymphocytes which are specifically combined with the KET-BSA conjugate are separated by a flow cytometry, the nucleotide sequences of heavy chains and light chains of the antibodies in the lymphocytes are amplified by single-cell PCR, the obtained sequences are constructed into a complete mouse IgG antibody expression vector, the monoclonal antibodies are expressed by transient HEK293F cells, and the monoclonal antibodies are purified. Compared with the traditional monoclonal antibody preparation, the time is greatly shortened, the obtained monoclonal antibody has high stability and good uniformity, and the batch-to-batch difference is greatly reduced.
The application comprises the following steps: (1) The spleen of the Balb/c mouse is taken after the Balb/c mouse is immunized for a plurality of times by using a KET-BSA conjugate, spleen lymphocytes are separated by using a lymphocyte separation liquid kit, B lymphocyte suspension is separated by using a BD FACS flow cytometer, the B lymphocyte suspension is collected in a 96-well PCR plate containing proper cell lysate, an RNase inhibitor and a PCR reaction reagent, a mixture of forward primers is designed for different leader sequences of a heavy chain variable region of an antibody, reverse primers are specifically complementary to an antibody constant region, mRNA is reversely transcribed into cDNA, the corresponding antibody variable region nucleotide sequence is cloned by RT-PCR, gel electrophoresis analysis, purification and sequencing are carried out, and finally the antibody nucleotide sequence capable of being combined with the KET-BSA conjugate is obtained.
(2) The heavy and light chain variable region sequences were constructed into complete murine IgG expression vectors and monoclonal antibodies were expressed using HEK293F cells, purified using Protein a affinity chromatography, and colloidal gold particles were labeled, respectively.
(3) The colloidal gold chromatography screening platform is utilized to display that the 4A1 monoclonal antibody and the 6B2 monoclonal antibody are two dominant monoclonal antibodies for detecting ketamine.
SEQ ID NO1: an anti-Ketamine (KET) specific antibody 4A1 light chain variable region amino acid sequence;
SEQ ID NO2: an anti-Ketamine (KET) specific antibody 4A1 heavy chain variable region amino acid sequence;
SEQ ID NO3: an anti-Ketamine (KET) specific antibody 6B2 light chain variable region amino acid sequence;
SEQ ID NO4: an anti-Ketamine (KET) specific antibody 6B2 heavy chain variable region amino acid sequence;
SEQ ID NO5: anti-Ketamine (KET) specific antibody 4A1 light chain variable region nucleotide sequence;
SEQ ID NO6: anti-Ketamine (KET) specific antibody 4A1 heavy chain variable region nucleotide sequence;
SEQ ID NO7: anti-Ketamine (KET) specific antibody 6B2 light chain variable region nucleotide sequence;
SEQ ID NO8: anti-Ketamine (KET) specific antibody 6B2 heavy chain variable region nucleotide sequence;
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sequence listing
<110> Hangzhou xian to Biotechnology Co., ltd
<120> anti-ketamine specific antibodies, plasmid vectors and methods
<130> 20211207
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Claims (7)
1. An anti-ketamine specific antibody comprising a light chain and a heavy chain, characterized in that:
the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 1;
the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.
2. The anti-ketamine-specific antibody of claim 1, wherein:
the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.1 is shown as SEQ ID NO. 5; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.2 is shown as SEQ ID NO. 6.
3. A plasmid vector combination, characterized in that: the plasmid vector combination contains nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6.
4. An anti-ketamine specific antibody comprising a light chain and a heavy chain, characterized in that:
the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 3;
the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 4.
5. The anti-ketamine-specific antibody of claim 4, wherein:
the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.3 is shown as SEQ ID NO. 7; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.4 is shown as SEQ ID NO. 8.
6. A plasmid vector combination, characterized in that: the plasmid vector combination contains nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO. 8.
7. A method of producing an anti-ketamine-specific antibody of any one of claims 1-2 and 4-5, comprising the steps of:
a) The nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region are respectively bridged with the nucleotide sequence of the mouse IgG light chain constant region and the nucleotide sequence of the heavy chain constant region by PCR, and then are respectively connected with plasmid vectors to construct eukaryotic cell expression vectors;
b) Transfecting the eukaryotic expression vector in the step a) to HEK293F cells to express to obtain an anti-Ketamine (KET) monoclonal antibody;
c) Purifying the monoclonal antibody, and determining dominant monoclonal antibody through a colloidal gold chromatography experiment;
the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.5 or as SEQ ID NO. 7;
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.6 or SEQ ID NO. 8.
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| CN113150136A (en) * | 2021-04-30 | 2021-07-23 | 杭州贤至生物科技有限公司 | Preparation of novel coronavirus N protein monoclonal antibody |
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